Systems, devices and methods for three-dimensional imaging of moving particles

ABSTRACT

Disclosed are methods, devices, systems and applications for camera-less, high-throughput three-dimensional imaging of particles in motion. In some aspects, a system includes a particle motion device to allow particles to move along a travel path; an optical illumination system to produce an asymmetric illumination area of light in a region of the travel path of a particle that scans over a plurality of sections of the particle at multiple time points while the particle is moving; an optical detection system optically interfaced with the particle motion device to obtain optical signal data associated with different parts of the particle corresponding to the particle&#39;s volume during motion in the travel path; and a data processing unit to process the optical signal data obtained by the optical detection system and produce data including information indicative of 3D features of the particle.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent document is a continuation of U.S. patent application Ser.No. 16/970,304, filed on Aug. 14, 2020, which is a national stageapplication of and claims the benefit of International Application No.PCT/US2019/018631, filed on Feb. 19, 2019, which claims priority to andbenefits of U.S. Provisional Patent Application No. 62/710,576, filed onFeb. 16, 2018. The entire content of the aforementioned patentapplications are incorporated by reference as part of the disclosure ofthis patent document.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under DA045460 awardedby the National Institutes of Health. The government has certain rightsin the invention.

TECHNICAL FIELD

This patent document relates to techniques and systems for flowcytometry.

BACKGROUND

Flow cytometry is a technique to detect and analyze particles, such asliving cells, as they flow through a fluid. For example, a flowcytometer device can be used to characterize physical and biochemicalproperties of cells and/or biochemical molecules or molecule clustersbased on their optical, electrical, acoustic, and/or magnetic responsesas they are interrogated by in a serial manner. Typically, flowcytometry uses an external light source to interrogate the particles,from which optical signals are detected caused by one or moreinteractions between the input light and the particles, such as forwardscattering, side scattering, and fluorescence. Properties measured byflow cytometry include a particle's relative size, granularity, and/orfluorescence intensity.

SUMMARY

Disclosed are methods, devices, systems and applications that pertain tothree-dimensional imaging flow cytometry (3D-IFC).

In some aspects, a system for three-dimensional (3D) imaging of movingparticles includes a particle motion device including a substrate toallow particles to move along a travel path in a first direction; anoptical illumination system to produce an asymmetric illumination areaof light in a region of the travel path of a particle that scans over aplurality of sections of the particle at multiple time points while theparticle is moving, the asymmetric illumination area of light includingone dimension of illumination thinner than the other dimension ofillumination to form a shape like an illumination plane, the opticalillumination system including a light source to produce a light beamthat is optically coupled to light redirection device to modify thelight beam by redirecting the light beam to different angles to have theasymmetric illumination area of light directed at the travel path ofparticle motion; an optical detection system optically interfaced withthe particle motion device and operable to obtain optical signal dataassociated with different parts of the particle corresponding to theparticle's volume during motion in the travel path, in which the opticaldetection system includes one or more photodetectors and a spatialfilter positioned between the particle motion device and the one or morephotodetectors, the spatial filter including a plurality of apertures toselectively allow a portion of the asymmetric illumination area of lightover a scanned section of the particle to pass through the spatialfilter and be detected by the one or more photodetectors; and a dataprocessing unit in communication with the optical detection system, thedata processing unit including a processor configured to process theoptical signal data obtained by the optical detection system and producedata including information indicative of 3D features of the particle.

In some aspects, a method for three-dimensional (3D) imaging of movingparticles includes moving a particle along a first direction; scanning aplurality of sections of the particle, section by section, by directingindividual asymmetric illumination areas of light at correspondingregions while the particle is moving; spatially filtering portions ofthe asymmetric illumination area of light over the scanned sections ofthe particle in motion to allow certain optical signals corresponding tothe particle's volume through a spatial filter to be detected; anddetecting the spatially-filtered optical signals to obtain individualvoxels in three dimensions mapped to time points of detection.

The subject matter described in this patent document and attachedappendices can be implemented in specific ways that provide one or moreof the following features.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a block diagram of an example embodiment of athree-dimensional imaging flow cytometry (3D-IFC) system in accordancewith the present technology.

FIG. 1B shows a block diagram of an example embodiment of the dataprocessing system of the 3D-IFC system shown in FIG. 1A.

FIG. 1C shows a diagram of an example embodiment of a 3D-IFC system inaccordance with the present technology.

FIG. 1D shows an illustration depicting a cell flowing at the objectplane and a spatial filter positioned at image plane from the diagram ofFIG. 1C.

FIGS. 2A-2D show diagrams illustrating the principle of the relationbetween time-domain signal and spatial information including spatiallight intensity.

FIGS. 3A and 3B show data plots of time-domain signals from an example3D-IFC system, in accordance with some example embodiments.

FIGS. 4A-4I show data plots of 2D and 3D images captured by an example3D-IFC system, in accordance with some example embodiments.

FIG. 5A shows a diagram of an example embodiment of a 3D-IFC system inaccordance with the present technology.

FIG. 5B shows an illustration depicting a cell flowing along the objectplane and a spatial filter positioned at image plane from the diagram ofFIG. 5A.

FIG. 5C shows an example of a 3D reconstructed space produced by thedata processing system of an example 3D IFC system.

FIGS. 5D-5F show an example output of an example 3D-IFC systemdemonstrating time to 3D-space mapping, in accordance with some exampleembodiments.

FIG. 6A-6C show images and data plots from an example 3D-IFC systemusing CFSE-stained HEK-293 cells bound with 1 μm fluorescent beads, inaccordance with some example embodiments.

FIGS. 7A and 7B show images and data plots of fluorescent γH2AX fociimaged by an example 3D-IFC system, in accordance with some exampleembodiments.

FIGS. 8A-8D show images and data plots depicting leukocyte cellmorphology determined by an example 3D-IFC system, in accordance withsome example embodiments.

FIG. 9 shows an example of a microfluidic system used in an example3D-IFC system, in accordance with some example embodiments.

FIG. 10 shows diagrams of examples for a spatial filter design, inaccordance with some example embodiments.

FIG. 11 shows a diagram of an example embodiment of a 3D-IFC system inaccordance with the present technology.

DETAILED DESCRIPTION

A central challenge of biology is to correlate the phenotype ofheterogeneous individuals in a population to their genotype in order tounderstand the extent to which they conform to the observed populationbehavior or stand out as exceptions that drive disease or the ability tobecome threats to health. While optical microscopy has been acornerstone method to study the morphology and molecular composition ofbiological specimens, flow cytometry has been a gold standard forquantitative high-throughput single-cell characterization in numerousbiomedical applications. Conventional imaging flow cytometry (IFC) is atechnique that merges some aspects of optical imaging with flowcytometry. For example, IFC can simultaneously produce ensemble-averagedmeasurements and high-content spatial metrics from individual cells in alarge population of cells, without perturbation due to experimentcondition change. Yet, an important limitation of existing IFC systemsis that, regardless of the optical detection method and computationalgorithm is used, only 2D cell images can be obtained. The absence of3D tomography results in occlusion of objects, blurring by focal depth,loss of z-axis spatial resolution, and artifacts due to projection of a3D cell into a 2D image. For example, with 2D microscopic imaging, if afluorescent probe is observed at the center of a cell, its location(e.g. membrane, cytosol, nucleus) is ambiguous. For a range ofapplications, such as internalization measurements, probeco-localization, and spot counting, relative to 2D imaging that isdependent on the cellular orientation to the imaging plane, 3D imagesprovide more complete and accurate phenotyping of cell and organellemorphology, as well as nucleic acid and protein localization to supportbiological insights. Thus, there is a need for rapid and continuous 3Dimage acquisition for single cells in flow.

The demand of continuous, sub-millisecond temporal resolution usingfluorescence microscopy remains largely unsatisfied. Imaging cells inflow can quickly perform high-throughput morphology, translocation andcell signaling analysis on a large population of cells. Compared tosingle-point flow cytometry, three-dimensional imaging flow cytometry(3D-IFC) provides high-content information about 3D spatial distributionof fluorescence and/or scattering light that can be useful forhigh-throughput rare cell detection and heterogeneous phenomena studies.

Disclosed are methods, devices, systems and applications forcamera-less, high-throughput three-dimensional imaging of particles inmotion. In some aspects, for example, a three-dimensional imaging flowcytometry (3D-IFC) system is arranged in a light sheet fluorescencemicroscopy configuration, which illuminates a specimen in a single planeat a time whilst the signal is detected in a perpendicular direction,e.g., through a spatial filter. For example, the 3D-IFC system caninclude an acousto-optic deflector providing high-speed scanning in thez-direction. For example, the 3D-IFC system can include a single-elementphotodetector to detect the fluorescence optical signal passed thespatial filter in each signal channel, and a 3D image is reconstructedfrom the time-domain detector output. The 3D-IFC system integrates thescanning light sheet, the cell's flow motion, and the spatial filter,such that the detector is able to detect an individual point in the cellat a time, which allows space-to-time mapping on a one-to-one basis, socalled spatial-to-temporal transformation.

While some of the disclosed embodiments of the systems, methods anddevices for three-dimensional imaging of moving particles are describedherein primarily for imaging flow cytometry to facilitate understandingof the underlying concepts, it is understood that the disclosedembodiments can also include techniques and systems for moving particlesby other means or modalities and be used for other applications.

The disclosed systems, methods and devices are able to map the 3Dfeatures of an object in motion (e.g., cell, particle or other object),voxel by voxel, to a temporal signal at the detector output when thecell/particle/object travels through (e.g., by flow, slide, fall, orother mechanism) an optical interrogation zone defined by an opticalillumination area, referred to the “light sheet”, that is fast scanning.In some embodiments, the disclosed systems, methods and devices includea spatial filter/mask that is placed in front of an optical detector toonly allow the optical signal generated from a specific volume (e.g.,point volume) and at a specific time to reach the detector(s). Signalsoutside the specific volume or specific time point are blocked by thespatial mask.

FIG. 1A shows a block diagram of an example embodiment of a system forthree-dimensional imaging of moving particles, labeled 100, inaccordance with the present technology. The system 100 includes aparticle motion system 110, a scanning light-sheet illumination system120 interfaced with the particle motion system 110, an optical detectionsystem 130 interfaced with the particle motion system 110, and a dataprocessing system 140 in communication with the optical detection system130 and/or scanning light-sheet illumination system 130. In someimplementations, the system 100 operates as a single point detector bytransforming the optically detected information about the specimen inflow between space and time to construct a three-dimensional image fromthe transformed information. For example, instead of focusing light on asingle spot of the specimen, the illumination system 120 focuses thelight along a 2D plane as the specimen is in motion by the particlemotion system 110, while the optical detection system 130 detectsindividual light points from the three-dimensional space around theplanarly-illuminated specimen based on a spatial filter map having apattern that allows only certain portions of the light to pass throughto limit certain point signals to enter the detector. Thisopto-mechanical technique structures light on a three-dimensional objectin two dimensions (e.g., a planar “light sheet”) while scanningoptically-screened one-dimensional points of the planar light sheetwhile the object is in motion. The system 100 is able to produce 3D celltomographic images of particles, cells or other moving objects at a fastflow rates (e.g., 10 cm/sec), allowing high-throughput 3D imaging.

In some embodiments of the system 100, the particle motion device 100includes a substrate that allows particles to move along a travel pathin a first direction (e.g., motion direction). The scanning light-sheetillumination system 120 is configured to produce the light sheet in aregion of the travel path of a particle that scans over a plurality ofsections of the particle at multiple time points while the particle ismoving. The produced, scanning light sheet is an asymmetric illuminationarea of light that includes one dimension of illumination that isthinner than the other dimension of illumination to form a shape like anillumination plane. The scanning light-sheet illumination system 120includes a light source (e.g., laser) to produce a light beam that isoptically coupled to light redirection device to modify the light beamby redirecting the light beam to different angles to have the lightsheet directed at the travel path of particle motion. The opticaldetection system 130 is optically interfaced with the particle motiondevice 110 and operable to obtain optical signal data associated withdifferent parts of the particle corresponding to the particle's volumeduring motion in the travel path. The optical detection system 130includes one or more photodetectors and a spatial filter positionedbetween the particle motion device 110 and the one or morephotodetectors, where the spatial filter include one or more aperturesto selectively allow a portion of the asymmetric illumination area oflight over a scanned section of the particle to pass through the spatialfilter and be detected by the one or more photodetectors. The dataprocessing unit 140 is in communication with the optical detectionsystem 130 (and, in some embodiments, with the scanning light-sheetillumination system 120) and configured to process the optical signaldata obtained by the optical detection system 130 and produce dataincluding information indicative of 3D features of the particle.

In some embodiments, the particle motion system 110, sometimes referredto as a “flow cell” in such related embodiments, includes a substratehaving a fluidic channel for carrying a fluid sample containing a flowspecimen (e.g., particles, living cells, or other objects). In someembodiments, the particle flow cell system includes a flow focusingsystem to produce a confined sample fluid to a fine stream in thefluidic channel. In some embodiments, the fluidic channel of the flowcell is configured in an interrogation region to be opticallytransparent, allowing for clear optical paths. The fluid containing theflow specimen flows along a flow direction through the interrogationarea in the fluidic channel, where optical data are obtained by theimaging system 120 for each particle, including single particles orsingle cells.

Yet, in some embodiments, the particle motion system 110 includes asubstrate to which one or more particles, cells (or other object forinterrogation) are fixed, where the substrate is moved with respect tothe scanning light-sheet illumination system 120 and optical detectionsystem 130 such that a particle, cell, etc. is brought into theinterrogation region upon which the scanning light sheet is illuminatedand from which such light at and/or through the particle, cell, etc. isdetectable. In such embodiments, the particle motion system 110 includesa device positioning system including a moving stage (e.g., driven by amotor or other driving device) to move the substrate such that theparticle fixed thereon is moved along the travel path and brought intothe region upon which the light sheet is scanned,

In some embodiments, the scanning light-sheet illumination system 120includes a light source to generate light that will be structured as thelight sheet for illumination on a particle flowing in the fluidicchannel of the particle motion system 110. In some embodiments, thelight source includes a laser; and/or in some embodiments, the lightsource includes a high-brightness LED or super-luminescent diode. Theillumination system 120 includes a device for redirecting the light beamgenerated by the light source to different angles, which can beprogrammed to generate periodic angle change at high speed (e.g., arange of at least 100 kHz to 1 MHz). The light beam redirection devicecan be optically coupled with passive optical components (e.g., such asa cylindrical lens or other optical components, like a lenses, prisms,or spatial filters) to shape the redirected light and produce thestructured light sheet illumination that allows optical sectioning ofthe particles in motion. For example, the illumination system 120 isoperable to illuminate a thin slice or a thin line of the particle at atime, e.g., a scanning light-sheet on the particle.

In some embodiments, for example, the light redirection device includesacousto-optic deflector (AOD) device. For example, the AOD can providefor controllable, high speed changing of the angle of the light beamwith high optical efficiency, where the AOD moves the energy of the beamwithout losing portions of the beam. While the AOD redirects lightreliably with high speed and efficiency, it is wavelength sensitive andmay not be optimal for some applications where multiple light beams ofvarying wavelengths are to be employed in the implementation of thesystem 100.

In addition to or alternative to the AOD, for example, in someembodiments, the light redirection device can include a multiplexedarray of scanning mirrors and/or multiplexed array of scanning gratings,which are not wavelength limited. For example, an individual opticalcomponent like a scanning mirror would provide scanning speeds of about20 kHz; but, the multiplexed array of scanning mirrors can be configuredto provide high speeds, e.g., 200 kHz.

In some implementations, the optical detection system 130 can be placedat any angle orthogonal to that of the travel path of the particle,cell, etc., e.g., such as normal to a fluidic channel of the particleflow cell that has an optically clear path. In some embodiments, theoptical detection system 130 includes a photodetector and a spatialfilter positioned at an image plane of the photodetector to selectivelyallow the light from specific positions of the particle to pass throughfor detection by the photodetector. The optical detection system 130 isoperable to detect 1D optical data points in time of a 3D objectionbased on combination of (a) flow motion of the particle, (b) a scanninglight-sheet illumination, and (c) spatially mapped filter to detectlight intensity (e.g., including fluorescence and scattering) from everyposition of the particle in a sequentially-timed manner. Therefore, thetime-domain signal recorded by photodetector(s) of the optical detectionsystem 130 has a one-to-one relation from space to time. In someimplementations, the optical detection system 130 includes opticalfilters in order to collect and detect fluorescence and scatteringlight. For example, the optical detection system 130 can include aplurality of photodetectors for detecting multiple channels offluorescence and/or scattering light.

In some embodiments, the spatial filter includes a surface having aplurality of apertures (openings or slits) arranged in a pattern on thesurface. In some embodiments, the slits are spaced apart along the firstdirection of particle motion and a second direction perpendicular to thefirst, where the position of the adjacent slits varies along the seconddirection with respect to another slit of the pattern. In some examples,the apertures can be configured in other geometries and shapes, e.g.,including but not limited to square, rectangular, trapezoidal,triangular, etc. or other curved shapes including oval, circular, etc.In various embodiments, the size of the apertures can be selected basedon the desired resolution of the image data to be constructed from thedetectable optical signals. For example, the diameter or length of theaperture can be determined from the magnification strength of the lightdirected onto the image plane multiplied by the desired resolution. Inone example, if the desired resolution is 1 μm and the objective lens ofthe detection module 170 is 20×, then the slit would be configured tohave a 20 μm diameter. Similarly, for example, if the desired resolutionis 0.5 μm and the objective lens of the detection module 170 is 50×,then the slit would be configured to have a 25 μm diameter. In someimplementations, for example, the relatively smaller apertures (withrespect to the particle size) effectively allow the detected light to beprocessed as a delta function.

The data processing system 140 is configured to process the detectedoptical signal data and produce data (e.g., a data set) that isindicative of three-dimensional features of the particle, cell, etc.from which a 3D image or density distribution of the particle, cell,etc. can be constructed. For example, the data may represent an opticaldensity distribution in 3D space, which is rather different than 3Dimages obtained from a conventional 3D imaging modality such as aconfocal microscope. In some embodiments, the data processing system 140includes a data streaming unit in communication with the opticaldetection system 130. The data processing system 140 can include aprocessor and memory (data processing unit) to reconstruct 3D image foreach particle from the detected optical signal data captured at theoptical detection system 130. In some implementations, for example, animage reconstruction algorithm is resident on the data processing unitthat includes specifications of illumination and detection protocols andcommands the scanning light-sheet illumination system 120 and theoptical detection system 130 accordingly. In some embodiments, the dataprocessing system 140 includes an image processing unit to process theimage data to determine properties associated with the particle.

FIG. 1B shows a block diagram of an example embodiment of the dataprocessing system 140 of the example system 100. In variousimplementations, the data processing system 140 is embodied on one ormore personal computing devices, e.g., including a desktop or laptopcomputer, one or more computing devices in a computer system orcommunication network accessible via the Internet (referred to as “thecloud”) including servers and/or databases in the cloud, and/or one ormore mobile computing devices, such as a smartphone, tablet, or wearablecomputer device including a smartwatch or smartglasses. The dataprocessing system 140 includes a processor to process data, and memoryin communication with the processor to store and/or buffer data. Forexample, the processor can include a central processing unit (CPU) or amicrocontroller unit (MCU). In some implementations, the processor caninclude a field-programmable gate-array (FPGA) or a graphics processingunit (GPU). For example, the memory can include and storeprocessor-executable code, which when executed by the processor,configures the data processing system 140 to perform various operations,e.g., such as receiving information, commands, and/or data, processinginformation and data, such as from the system 100, and transmitting orproviding processed information/data to another device, such as anactuator or external display. To support various functions of the dataprocessing system 140, the memory can store information and data, suchas instructions, software, values, images, and other data processed orreferenced by the processor. For example, various types of Random AccessMemory (RAM) devices, Read Only Memory (ROM) devices, Flash Memorydevices, and other suitable storage media can be used to implementstorage functions of the memory. In some implementations, the dataprocessing system 140 includes an input/output (I/O) unit to interfacethe processor and/or memory to other modules, units or devices. In someembodiments, such as for mobile computing devices, the data processingsystem 140 includes a wireless communications unit, e.g., such as atransmitter (Tx) or a transmitter/receiver (Tx/Rx) unit. For example, insuch embodiments, the I/O unit can interface the processor and memorywith the wireless communications unit, e.g., to utilize various types ofwireless interfaces compatible with typical data communicationstandards, which can be used in communications of the data processingsystem 140 with other devices, e.g., such as between the one or morecomputers in the cloud and the user device. The data communicationstandards include, but are not limited to, Bluetooth, Bluetooth lowenergy (BLE), Zigbee, IEEE 802.11, Wireless Local Area Network (WLAN),Wireless Personal Area Network (WPAN), Wireless Wide Area Network(WWAN), WiMAX, IEEE 802.16 (Worldwide Interoperability for MicrowaveAccess (WiMAX)), 3G/4G/LTE/5G cellular communication methods, andparallel interfaces. In some implementations, the data processing system140 can interface with other devices using a wired connection via theI/O unit. The data processing system 140 can also interface with otherexternal interfaces, sources of data storage, and/or visual or audiodisplay devices, etc. to retrieve and transfer data and information thatcan be processed by the processor, stored in the memory, or exhibited onan output unit of a display device or an external device.

FIG. 1C shows a diagram of an example embodiment of a 3D-IFC system 150in accordance with some embodiments of the system 100. As shown in thediagram, the 3D-IFC system 150 includes a particle flow cell 151 thatincludes a fluidic channel for carrying a fluid sample containing a flowspecimen (e.g., particles, living cells, etc.) that is interfaced to alight sheet illumination module 160 and a detector module 170. The lightsheet illumination module 160 includes an acousto-optic deflector (AOD)165 optically coupled to a cylindrical lens 161 and an objective lens(OL) 163, arranged in an optical illumination path with a light source169 (e.g., a laser, such as a 488 nm laser). For example, thecylindrical lens 161 is placed after the AOD 165 to focus the light(e.g., make the laser beam focus) in one direction to form a scanninglight sheet. The AOD produces deflected first-order beam at a differentangle for each frequency and therefore generates scanning over time. Thedetector module 170, which includes a spatial filter (SP) 171, arrangedin an optical detection path between an objective lens 172 interfacedwith the flow system 151 and an optical photodetector 179 (e.g.,photomultiplier tube (PMT)) to capture the optical signal data from theflow objects (e.g., cells, particles, etc.). In some implementations, aparticular light spectral range or ranges of the optical signal data isdetected by the detector module 170. As such, in some embodiments likethat shown in FIG. 1C, for example, the detector module 170 includes thespatial filter 171 optically coupled to a dichroic mirror (DM) 173 thatis optically coupled to a bandpass filter (BP) 175 and the photodetector179 (e.g., PMT) in split optical paths. For example, the illuminationcan be done perpendicularly to the detection. The spatial filter 171 isplaced at the image plane. In some embodiments, the 3D-IFC system 150includes lenses or light guides to focus and/or direct light at thedesignated location of another component of the system. In someimplementations, for example, PMT signals are collected, e.g., by 100Mega samples per second (MS/s) per channel digital recording device.

In some example embodiments, the 3D-IFC system 150 is arranged in alight sheet fluorescence microscopy configuration, which illuminates aflow specimen in a single plane at a time whilst the signal is detectedin a perpendicular direction. The acousto-optic deflector (AOD) 165 canprovide the high-speed scanning in z-direction. A single-elementphotodetector, such as PMT 179, detects fluorescence passed the spatialfilter 171 in each channel, and an image is reconstructed from thetime-domain detector output. Combining the scanning light sheet, thecell's flow motion, and the spatial filter, the detector only detects anindividual point in the cell at a time, which allows spatial-to-temporaltransformation.

FIG. 1D shows an illustration depicting an example flow specimen (e.g.,a cell) flowing along the object plane and spatial filter 171 positionedat image plane. In some embodiments, the spatial filter 171 includes asurface having a plurality of apertures (openings) arranged in apattern, e.g., a slit, where each aperture of the pattern is spacedapart along the y-axis (parallel to the flow direction) and varies inits position along the x-axis (perpendicular to the flow direction) withrespect to another aperture of the pattern, such that each aperturealong the y-axis is at a different position along the x-axis. In theexample spatial filter 171 shown in FIG. 1D, the apertures include slitshaving a rounded shape (e.g., oval or circular) with a diameter of about20 μm.

As shown in the diagram, the planar light sheet is created in the x-yplane, where the width of light is defined along the y-axis (theparticle flow direction), and where the x-axis is the weight of thelight (the direction of illumination). In this diagram, the z-axis isthe scanning axis, which is controlled by the AOD 165. In someimplementations, the thickness of a light sheet (z-axis) is on the orderof about 1 μm. The optical point scanning of the cell is carried outalong the y-axis while the cell is in motion in the channel; and thex-axis scanning is accomplished by the spatial filter map 171. In thisexample, the light sheet produced by the light sheet illumination module160 has a 0.6 μm waist (z-direction) and 250 μm length in y-direction,scanning in z-direction. The spatial filter has ten 20 μm (x-direction)by 10 μm (y-direction) slits positioned apart in the way of one isimmediately after another.

FIGS. 2A-2D show diagrams illustrating a relationship between atime-domain signal (right) and spatial information (left) depicted as acube in 3D space, showing the spatial light intensity. FIG. 2A shows anexample of the time-domain signal from 3D-IFC represents the lightintensity of a 3D object (sphere-like in the example); each time-pointcorresponds to a specific voxel in the 3D space. FIG. 2B shows anexample of an envelope of the signal which represents the xy-planeprojection of the whole 3D object. FIG. 2C shows an example in whicheach section as highlighted represents the light intensity of oneyz-plane slice of the 3D space. In these examples, the time duration ofeach section is the time for the object to travel 20 μm in the objectplane, which equals to the image passes one slit to another along thespatial filter. FIG. 2D shows an example in which one single peak ashighlighted represents the light intensity of one row in z-direction,where the light sheet does one scan period.

The following equations may be used to describe some aspects of theembodiments of the system 100. For example, when the acoustic frequencysent to the acoustic transducer in the acousto-optic deflector (AOD) ischanging in a sinusoidal manner, the position of the light sheet inz-direction f (t) can be described as

f(t∈(0,T/2))=v ₁ t

f(t∈(T/2,T))=2z ₀ −v ₁ t  (1)

For example, if the acoustic frequency is changing in a sawtooth-wavemanner, f (t) can be described as

f(t)=v ₁ t,  (2)

where T is the scanning period, v₁ is the scanning speed in z-direction,z₀ is the waist of the light sheet in z-direction.

The light sheet illumination I(x, y, z, t) can be written as

$\begin{matrix}{{I\left( {x,y,z,t} \right)} = {{C \cdot e^{- \frac{{({z - {f(t)}})}^{2}}{\sigma^{2}}}} \approx {C \cdot {\delta\left( {z - {f(t)}} \right)}}}} & (3)\end{matrix}$

The characteristic function F (x, y) of the spatial filter is designedto be

F(x,y)=Σ_(q=1) ^(N)δ(x−q)·δ(y−qL),  (4)

where x=1, 2, . . . , N is the number of slits on the spatial filter, Lis the length of the slit in y-direction.

The measured PMT signal S(t) can be expressed as

S(t)=∫_(x,y,z)Cell(x,y−Mv ₂ t,z)I(x,y,z,t)F(x,y)dxdydz=∫_(x,y)Cell(x,y−Mv ₂ t,f(t))F(x,y)dxdy,  (5)

where M is the magnification factor of the detection system, Cell (x, y,z) is the 3D cell fluorescence intensity profile.

This example approach maps the 3D cell image into the time-domain lightintensity on a one-to-one basis.

Implementations of the example embodiments of the system 100 may be usedfor imaging and characterizing features of biological samples, such asliving cells. For example, single cells may be imaged in suspensionflowing at a speed of 0.1 m/s inside a flow-cell cuvette. In exampleimplementations described herein, some single-cell samples that havebeen imaged include HEK293T cell stained with CellTrace CFSE with 1 μmfluorescence beads bond to its cell membrane.

FIGS. 3A and 3B show example of data plots of time-domain signals fromthe example 3D-IFC system 150. The object, an HEK293T cell stained withCellTrace CFSE with 1 μm fluorescence beads bond to its cell membrane,may be flowing at speed of 0.1 m/s. The data plots show the time-domaindata output from the PMT of one channel (e.g., 520/40 nm bandpass) inFIG. 3A, and another channel (e.g., 650 nm longpass) in FIG. 3B. Theenvelopes detected may be used to reconstructed the 2D image of thecell. The data plots of FIGS. 3A and 3B show time-domain signal crops ofa cell as an example taken with the 3D-IFC system 150. In these exampleimplementations, the light sheet was scanning at 200 kHz within a rangeof 25 μm in z-direction.

FIGS. 4A-4I show example data plots of 2D images and 3D images capturedby the example 3D-IFC system 150 of the HEK293T cell stained withCellTrace CFSE with 1 μm fluorescence beads bond to its cell membrane.In FIGS. 4A-4C, the data plots show 2D images showing green channel(401), red channel (402), and overlay (403) image data, respectively,which were captured and reconstructed by the example 3D-IFC system 150.In these data plots, the scale bars represent 5 μm. In FIGS. 4D-4F, thedata plots show 3D reconstructed images showing green channel (404), redchannel (405), and merged (406) 3D reconstructed image data produced bythe example 3D-IFC system 150. In these data plots, the cube cagerepresents a 20 μm×20 μm×20 μm space. FIGS. 4G-4I show six different 2Dperspectives of the 3D imaged cell in the green channel (perspectives407-411), the red channel (perspectives 412-417) and the overlay(perspectives 418-423) for 3D images. As shown by the 3D reconstructiveimages, the 2D perspectives from different viewing angles possessdifferent features.

The 3D images shown in FIGS. 4A-4I demonstrate that there are fourfluorescence beads on the cell membrane, but the 2D images containsinexplicit information about the number of beads. In a flow system, onecan hardly control the orientation of an object, so capturing 2D imagesthat have only one viewing perspective can generate detection error,especially when relative location is important. As biological specimensare intrinsically three dimensional, capturing 3D images providescomplete information about the sample.

Example methods for implementing and/or fabricating the example 3D-IFCsystem 150 are described. In the example implementations describedabove, a 100 mW 488 nm laser (iBeam-SMART, Toptica) was used that has anoval beam shape with Gaussian energy distribution, which is collimated,focused, and then expanded to illuminates an area of 1.2 μm(z-direction) by 250 μm (y-direction). An AOD (Isomet) that is driven bya modular, swept-frequency RF power source (Isomet) was used to producedeflected laser beam with different output angles at different time, andtherefore scan the laser beam in z-direction. A cylindrical lens and a10×/0.25 objective lens was used to form light sheet. The fluorescenceemission from the sample was collected through a 10×/0.25 objective lens(Mitutoyo) and detected by one PMT (Hamamatsu) at each channel. PMTreadouts were recorded by PCIE DAQ card (Advantech).

The fabrication of the spatial filter can include the follow techniques.The design of spatial filter was drawn in AutoCAD and printed to atransparency mask at 20,000 dots per inch (dpi). A layer of negativephotoresist (NR9-1500PY, Futurrex, Inc.) was spun at 3,000 rotations perminute (rpm) on a 6-inch glass wafer. The wafer was heated on a hotplate at 150° C. for 3 minutes then exposed to UV light (EVG62ONT, EVGroup) through the transparency mask. Post UV exposure, the wafer wasbaked at 100° C. for another 3 minutes before development in RD6(Futurrex, Inc.) for 12 seconds. A film of 300 nm thick aluminum wassputtered onto the glass wafer. After metal lift-off, the patterns ofthe spatial filter were formed and the glass wafer was diced into 12 mmby 12 mm pieces. To help hold the spatial filter in the flow cytometersystem, the spatial filter having ten 20 μm by 10 μm slits was mountedto a sample holder fabricated by 3D printing method.

Preparation of cell samples can include the following techniques. TheHEK293T human embryonic kidney cell samples were cultured with culturemedia (DMEM, 10% Fetal Bovine Serum, 1% Penicillin Streptomycin) to 90%confluency in 10 cm petri dish. After 100× dilution of the 1.0 μmfluorescent beads (Ex/Em: 488/645 nm, T8883, Thermo Fisher) from thestock solution, 100 μL of the diluted solution was mixed with 10 mLfresh cell culturing media and added to cell culturing plate. Aftercontinuous culturing for 10 hours, the HEK293T cells were harvested,fixed by 4% paraformaldehyde, washed and resuspended in 1× phosphatebuffered saline (PBS). After fixation, the cells were stained withCellTrace CFSE cell proliferation kit (Ex/Em: 492/517 nm, ThermoFisher). Before every imaging experiment, the suspension was diluted inPBS to a concentration of 200 cells/μL.

Example applications of the disclosed 3D-IFC systems and methods caninclude the following.

DNA Damage: When ionizing radiation or cytotoxic chemical agents causeDNA damage, which forms double stranded breaks (DSBs), thephosphorylated protein gamma-H2AX foci quickly form and represent theDSBs in a 1:1 manner. With an antibody raised against gamma-H2AX, thedetection and visualization of gamma-H2AX by flow cytometry andimmunofluorescence microscopy evaluate DNA damage, related DNA damageproteins and DNA repair. 2D imaging techniques and manual quantificationare widely used by many researchers; yet, counting number of foci from a2D image is not only extremely labor intensive but also unreliable whenthe number of foci is large and overlapping occurs in one perspective.Obtaining 3D spatial resolution at a high throughput is significantlysuperior for the analysis of the number, area and density of gamma-H2AXcomparing to the 2D techniques. The example embodiments of the 3D-IFCsystem 150 described herein can provide a high throughput cytometricinstrument that is suitable for gamma-H2AZ foci quantification, forexample.

FISH: Fluorescent in situ hybridization (FISH) can effectively detectand localize the presence or absence of specific DNA sequences onchromosomes and achieve high accuracy in disease diagnosis and prognosisassessment. FISH signal analysis is typically done on 2D microscopicimages. However, detection error may occur when two probed FISH signalsare overlapped in the projected image plane where the translocation isin the depth direction. For early or subtle diseases, detecting a smallnumber of abnormal cells can make diagnostic difference, this kind ofdetection error can be critical. The example embodiments of the 3D-IFCsystem 150 described herein may be suitable for 3D visualization of FISHsignals and may improve the overall accuracy of analysis for someclinical cases.

Internalization/Endocytosis: Many cellular processes such as, e.g., (1)phagocytes recognize, bind, internalize, and eliminate pathogen and celldebris; (2) cellular antibody uptake and presentation; (3) cellularexosome and other nanoparticle uptake; (4) cellular drug uptake andprocessing, are evaluated by measuring internalization. Either 2Dwide-field microscopic images or imaging cytometric images only providesincomplete information on the relative location. For example, from a 2Dmicroscopic image, if a particle is observed sitting right in the centerof a cell, one can hardly tell if the particle is on the cell membrane(on top of the cell) or the particle is inside the cytoplasm or even inthe cell nucleus. Instead, people take images at multiple focal planesto resolve this type of problem. The example embodiments of the 3D-IFCsystem 150 described herein can perform continuous high-throughputanalysis for these internalization studies.

Further embodiments and implementations of the cameraless,high-throughput 3D-IFC systems, methods and devices are described forimaging objects, like cells, based on optical sectioning microscopycaptures 3D fluorescence and darkfield images of single cells in flow.The disclosed cameraless, high-throughput 3D-IFC systems and methodsprovide the capability of co-capturing 3D fluorescence and label-freeside-scattering images of single cells in flow with a throughput ofapproximately 500 cells per second

For example, in some example implementations, a high-throughput 3D-IFCsystem utilizes light-sheet scanning illumination technique andspatial-to-temporal transformation detection technique to enablefluorescent and label-free 3D cell image reconstruction fromsingle-element photodetector readout without a camera. In the exampleimplementations, using the speed and sensitivity benefits ofphotomultiplier tubes (PMT), the example 3D-IFC system uses multiplescanning techniques to add spatial information in a flow cytometryarchitecture. 3D imaging may be achieved by laser scanning across thefirst (z-) axis, the cell translating by flow across the second (y-)axis, and the use of multiple pinholes arranged along the third (x-)axis to produce fluorescent and label-free information from 60,000voxels per cell. By precisely mapping time to space, a photodetectorreadout at one timepoint corresponds to one voxel in a 3D space. Exampleresults described below illustrate 3D-IFC of fluorescence and 90-degreelabel-free side-scattering (SSC) imaging of single cells in flow at avelocity of 0.2 m s−1, corresponding to a throughput of approximately500 cells per second.

FIG. 5A shows another example embodiment of a 3D-IFC system, labeled550, in accordance with some embodiments of the system 100. As shown inthe diagram, the 3D-IFC system 550 includes a flow system 551 thatincludes a fluidic channel for carrying a fluid sample containing a flowspecimen (e.g., particles, living cells, etc.) that is interfaced to alight sheet illumination module 560 and a detector module 570 of the 3DIFC system 550. The light sheet illumination module 560 includes anacousto-optic deflector (AOD) 565 optically coupled to a cylindricallens (CL) 561 and an illumination objective lens (10) 563 (e.g.,50×/0.55 illumination objective lens), arranged in an opticalillumination path with a light source 569 (e.g., a laser). Thecylindrical lens 561 is placed after the AOD 565 to focus the light(e.g., make the laser beam focus) in one direction to form a scanninglight sheet. For example, in some embodiments, the light sheetillumination module 560 includes a mirror element 564 between thecylindrical lens 561 and the AOD 565 to allow various positionalarrangements of the components of the illumination module 560 whilepreserving the optical path to deliver light on the flow sample. In someimplementations, the AOD 565 produces deflected first-order beam at adifferent angle for each frequency and therefore generates scanning overtime. In some implementations, the fluid sample is 2D hydrodynamicallyfocused by a sheath before entering the square cross section quartz flowcell, discussed further with respect to FIG. 9.

The detector module 570 includes a spatial filter (SP) 571, arranged inan optical detection path between a detector objective lens (DO) 572(e.g., 10×/0.28 detection objective lens) interfaced with the flowsystem 551 and an optical photodetector 579 to capture the opticalsignal data from the flow objects (e.g., cells, particles, etc.). Thespatial filter 571 is placed at the image plane of the photodetector579. For example, the photodetector 579 can be configured to include oneor more photodetectors (e.g., such as photomultiplier tubes (PMTs)) suchthat the detector module 570 detects a particular light spectral rangeor ranges of the optical signal data. As shown in FIG. 5A, in someembodiments, for example, the detector module 570 includes the spatialfilter 571 optically coupled to one or more dichroic mirrors (DM) 573that is optically coupled to corresponding photodetectors 579 (e.g.,PMT) in split optical paths. In some embodiments, the system 550 isconfigured to the one or more dichroic mirrors (DM) 573 opticallycoupled to corresponding bandpass filters (BP) 175 in split opticalpaths with the photodetectors 579. As shown in the diagram of FIG. 5A,for example, the illumination of the flow specimen is configured to beperpendicular to the detection orientation. In some embodiments, the3D-IFC system 550 includes lenses or light guides to focus and/or directlight at the designated location of another component of the system.

The 3D-IFC system 550 includes an example embodiment of the dataprocessing system 140, which includes a processing unit (e.g.,comprising a processor and memory) interfaced with a digitizer (DIG)580, e.g., 125 MS s⁻¹ digitizer, that digitally processes the outputsignals of the example PMTs of the detector module 570.

In example implementations of the 3D-IFC system 550, suspended cells mayform 2D hydrodynamically focused single file in a quartz flow cell witha square cross section, shown in further detail in FIG. 9. In theexample of FIG. 5A, a light-sheet (x-y plane) on the flow cell 551,e.g., with a diffraction limited beam waist and a height of 200 to 400μm, is formed by laser excitation, scanning in z-direction at a veryhigh rate (e.g., 200 kHz). For example, when a particle or cell flowingthrough the whole optical interrogation at 0.2 m s⁻¹, a pixelated fieldof view is represented by a 3D space with X by Y by Z voxels, as shownin FIG. 5C.

FIG. 5B shows an illustration depicting the optical interrogation areaof an example flow specimen (e.g., a cell) flowing along the objectplane and spatial filter 571 positioned at image plane. The spatialfilter 571 includes a surface having a plurality of apertures arrangedin a pattern, where each aperture of the pattern is spaced apart alongthe y-axis (parallel to the flow direction) and varies in its positionalong the x-axis (perpendicular to the flow direction) with respect toanother aperture of the pattern. As shown in the diagram of FIG. 5B, Hrepresents the height of the light-sheet; represents the tilt anglebetween flow (y-axis) and vertical line. Illumination light-sheetpropagates horizontally and scans in z-axis, sample flows in y-axis, xis the orthogonal axis.

The spatial filter at the image plane uses pinholes to produce linescans across the x-axis. For example, a pinhole array on the spatialfilter is aligned at a tilting angle, to the flow stream, so the pinholearray also steps along x-direction. In this manner, each pinhole allowslight from voxels with a distinct x-index to pass to PMT detector (e.g.,details of this example mask are discussed later in this disclosure).The imaging process begins when a flowing cell appears at the firstpinhole of the spatial filter. During the first light-sheet scanningperiod (e.g., 5 μs), light intensity of voxels z_(1-Z) with x₁y₁ indexis collected. As the cell flows downstream in y to the next position,x₁y₂, the corresponding z_(1-Z) voxels are produced. This is repeateduntil the cell completely passes pinhole 1 when the whole 2D yz-slice atx₁ is imaged. As the cell travels farther downstream in the y direction(e.g., flow direction), it reaches the following pinholes and yz-slicesof at x₂ to x_(X) are recorded. See FIG. 5C, for example.

FIG. 5C shows an example of a 3D reconstructed space produced by thedata processing system of the 3D IFC system 550. From the processing,for example, the resolution on the X-axis is determined by the number ofpinholes (e.g., pixelated field of view in x-direction); the resolutionof Y-axis is determined by the distance between two slits (e.g.,pixelated field of view in y-direction); and the resolution of Z-axis isdetermined by the light-sheet scanning range (e.g., pixelated field ofview in z-direction).

FIGS. 5D-5F show an example output of the 3D-IFC system 550. In theexample implementations of the system 550, the detected time-domainmulti-parametric signals (e.g., multi-color fluorescence, FL1 and FL2,and side-scattering, SSC, light intensities) were synchronized with thereference output of the tuning voltage of the AOD driver, which denotesthe z-position of the detected voxel. For example, at a 200 kHz scanningrate, a one-dimensional (1D) intensity profile in z-axis is recoveredfrom the time-domain signal within a time period of 5 μs (e.g., see FIG.5D). During the ˜100 μs of cell travel between pinholes, ˜20 periods oflaser scanning are performed, and an yz-plane 2D image array isrecovered from the PMT readout (e.g., see FIG. 5E). As the cell travelsthrough the entire interrogation area, a stack of 2D yz-plane images arerecovered, and the final 3D image is reconstructed, with the ordinalpinhole number indicating the voxel's x-position. In the example shownin FIG. 5F, a 10-pinhole spatial filter produces ten 2D yz-plane imagesand a signal length of 1 ms, corresponding to a throughput of 500 cellsper second. The bandwidth of the PMT and the digitizer used in theexample system 550, e.g., 150 MHz and 125 MHz, respectively, in thisimplementation, supports the throughput at 60,000 voxels per 3D cellimage. Each 3D cell image in a 3D space of 20 μm by 20 μm by 20 μm, isresized to 100 by 100 by 100 pixels for 3D image analysis andquantitative measurements.

To measure the speed of each cell for image reconstruction by thespatial-to-temporal transformation, pairs of slits upstream anddownstream of the pinholes are added to the optical spatial filter (see,FIG. 10). The measured rate of the cell moving through the detectionzone, and the known frequency of the light-sheet scanning rate ensureseach voxel in a 3D-IFC image has a distinct time-domain value, and allvoxels can be discreetly captured in time from single-element PMT. 3Dcell images are then reconstructed from the time-domain signal.

FIG. 5D depicts a one light-sheet scan period that produces 1D lightintensity profile in z-axis. The PMT voltage readout of one timepointcorresponds to the light intensity of one voxel in z-axis.

FIG. 5E depicts that while an object travels along y-axis, multiplescans produce a 2D profile in yz-plane within one pinhole scan period.Each section—separated by dotted lines—corresponds to the lightintensity of one row in the 2D image stack.

FIG. 5F depicts when an object completely passes through the spatialfilter covering area, the time-domain signal contains the completeinformation of the 3D profile in xyz-space. Each section corresponds toone 2D image slice. In the diagram of FIG. 5F, AOD refers to the tuningvoltage of the AOD driver; FL1 refers to the PMT readout of fluorescencedetection channel 1; FL2 refers to the PMT readout of fluorescencedetection channel 2; SSC refers to the PMT readout of side-scatteringlight detection channel.

To demonstrate the cellular imaging capability of 3D-IFC system 550, forexample, suspended single cells were imaged at a flow speed of 0.2 ms⁻¹. The example implementation of the system 550 produced example imagedata of mammalian cells, including two-color fluorescence and unlabeleddark-field (side-scattering) 3D images. For the example implementations,HEK293 cells were stained with an intracellular carboxyfluorescein dye(CFSE) and bound with a random number of 1 μm fluorescentcarboxylate-modified polystyrene beads.

FIG. 6A-6C show images and data plots from an example implementation ofthe 3D-IFC system 550 using CFSE-stained HEK-293 cells bound with 1 μmfluorescent beads. FIG. 6A depicts an example of recovered 2D yz-planeimages and the assembled 3D surface-rendered view of CFSE fluorescence,bead, and SSC (bottom row). FIG. 6B depicts examples of 3D images ofcells bound with beads and histogram detection events. The relativeposition relationship in 3D space indicates that the particle countingin the 3D-IFC is independent of cell orientation. In the example of cellbound with four beads, occlusion in specific perspective is a likelysource of error for particle counting with 2D images. FIG. 6C depicts anexample of intensity-based processing of 3D SSC images. The left columnof FIG. 6C shows intensity histograms of 3D SSC image of the cell shownin FIG. 7A. P(x, y, z) is the position of 1 μm size bead determinedusing 3D fluorescent image; within each bead position's ±1 μm area, thelocal intensity peak in 3D SSC image can be found. In FIG. 6C, the scalebars represent 5 μm. In the example implementations, the flow speed was0.2 m/s. The CFSE was intracellular carboxyfluorescein dye, with Ex/Em:488/517; the bead was carboxylate-modified fluorescent microspheres,with Ex/Em: 488/645; and the SSC, 90-degree side-scattering.

Referring to FIG. 6B, it is noted that while 2D bead images overlapped,the 3D-IFC resolved the exact number of particles from the reconstructed3D images, which is important for some localizing and co-localizingfeatures. In a flow system, cells and their internal structures areorientated at random, and as a result, 2D images may be from anunfavorable viewing perspective. Yet, using the disclosed 3D-IFCsystems, multiple perspectives can be achieved via 3D-IFC celltomography, which can provide improved relative position relationshipsand spot counting results, which favors both machine vision and humanvisualization. Using the side-scatter dark-field imaging mode, forexample, the 3D-IFC system 550 generated a 3D spatial distribution ofscattered light. Refractive index variations will scatter light when theobject is illuminated by visible light, and the size and refractiveindex distributions of the scattering regions determine thedistributions of the scattered light. The 3D SSC images represent thespatial distribution of those refractive index (n) variations among thefluid (PBS, n˜1.335), the cells (n˜1.3-1.6) and the polystyrene beads(n˜1.6). As shown in FIG. 6C, intensity-based low-pass filtered SSCimage indicates cell volume, and high-pass filtered SSC image correlateswith the fluorescent image of beads.

The disclosed 3D-IFC systems are capable of obtaining informationpractically unobtainable by conventional imaging flow systems. Take forexample the following. When ionizing radiation or cytotoxic chemicalagents cause DNA damage in the form of double stranded breaks (DSBs),the phosphorylated protein gamma-H2AX (γH2AX) forms foci at DSBs in a1:1 manner. With anti-γH2AX immunolabeling, foci reflect DNA damage andability for DNA repair. 2D imaging techniques and manual quantificationare used today, but counting foci from 2D images is labor intensive andunreliable due to perspective dependence. To evaluate foci counting ofthe 3D-IFC system, immunolabeled γH2AX foci in CMK3 cells may be imaged(e.g., a glioblastoma multiforme cell line) after 6 Gy ofgamma-irradiation. Representative cell images are shown in FIGS. 7A and7B. This example data show that the number of γH2AX foci is unrelated tothe fluorescence intensity, thus intensity-based measurements withconventional flow cytometry metrics poorly reflects DNA damage. Incontrast, the 3D-IFC system 550 was able to produce an accurate andrapid analysis of the number of γH2AX-positive foci.

FIGS. 7A and 7B show images and a data plot depicting example results offluorescent γH2AX foci imaged by the 3D-IFC system 550. FIG. 7A showsrepresentative 3D images of irradiation damaged glioblastoma CMK3 cellsstained with CFSE and γH2AX antibody conjugated PerCP/Cy5.5; and theirtwo-color fluorescence 2D yz-plane merged image slices at x=10 μm. Thehigh quality of the 3D images shows that the 3D-IFC is suitable forDNA-damage foci related study. FIG. 7B shows an example of a scatterplotof 917 detection events in the γH2AX intensity and foci count togetherwith images of the cells within the marked regions (i)-(iv) in thescatterplot. The data show that foci count is unrelated to thefluorescence intensity from labelled γH2AX, thus intensity-basedmeasurements with conventional flow cytometry metrics are unable toevaluate the extent of DNA damage. In the images, the scale barsrepresent 5 μm. The flow speed during the example implementations was0.2 m/s. For the CFSE, intracellular carboxyfluorescein dye, the Ex/Emwas: 488/517; for the PerCP-Cy5.5: DNA-damage antibody conjugated dye,the Ex/Em was: 490/677.

Peripheral blood leukocyte morphology is important clinical diagnosticand prognostic measure for acute and longitudinal evaluations. Exampleimplementations of the 3D-IFC system 550 were conducted to demonstratesuch peripheral blood leukocyte morphology.

FIGS. 8A-8D show images and data plots depicting leukocyte cellmorphology determined by the 3D-IFC system 550. FIG. 8A showsrepresentative 2D transmission images (left column) and 3D images ofleukocytes. FIG. 8B shows an example of an intensity histogram of SSCsignal of the first cell shown in FIG. 8A and its 3D profile inOrthoslice view and Volume view. Cell volume estimation based on 3D SSCimage matches the fluorescence volume. FIG. 8C shows a scatterplot of589 detection events in the CFSE (FL1) volume and SSC-based estimatedcell volume. FIG. 8D shows a scatterplot in the cell diameter calculatedfrom SSC-based estimated cell volume and SSC intensity. In thesefigures, the scale bars represent 5 μm. The flow speed was 0.2 m/s forthe example implementation.

FIG. 8A shows representative 3D-IFC imaging of leukocytes in threeimaging modes: 2D transmission image (e.g., a 3D-IFC parameter) and 3Dfluorescence and side-scattering images. The leukocyte SSC signal notonly indicates nuclear granularity but also provides cell volumetryobtained from low intensity (low refractive index) SSC imaging. Here twoor more intensity bands of 3D SSC signal can be used to generate two ormore images that reflect their corresponding refractive indices, such asthe cytosol and nucleus; whereas conventional flow SSC intensity signalis dominated by contributions of the nucleus. As shown in FIGS. 8B and8C, a 3D SSC image can be used to produce cell volume that matches thetransmission and fluorescence results.

Further information about the materials and methods used in the exampleimplementations of the 3D-IFC system 550 to obtain and produce theresults is described below.

In some embodiments, the particle motion system 110 (e.g., a particleflow cell embodiments) can include a microfluidic system like that shownin FIG. 9, which can continuously introduce suspended cells to theoptical interrogation area. Since the position of the cells in thecross-section of the microchannel is aligned with the optical field ofview in all the x-, y- and z-directions, a reproducible and stable flowspeed for cells is obtained by tightly focusing the cells at the centerof the square cross-section. Suspended cells in a sample injected by asyringe pump are hydrodynamically focused into a single stream. A sheathflow is used to confine flowing cells in both x- and z-direction. An airpressure pump together with a liquid flow meter are used to providestable sheath flow. At the junction of sheath flow and sample flow,tubing ends are specially tapered to keep symmetric flow. The flow rateratio between the sample and sheath is precisely controlled to be 100:1to ensure particles flowing at a high speed and within the optical fieldof view.

FIG. 9 shows an example of a microfluidic system 900 used for theexample particle flow cell 551 of the example 3D-IFC system 550. FIG. 9includes diagram and photograph of the example microfluidic system 900interfaced with tapered tubing. As shown in the diagram, suspendedparticles or cells in the fluid can flow through the channel to anexample quartz flow cell, which in this example has a cross-sectiondimension of 250 μm by 250 μm and a length of 20 mm.

In some example embodiments of the system 100, the optical system isarranged in a light sheet fluorescence microscopy configuration (likethat shown in FIG. 1C or FIG. 5A), which illuminates a specimen in asingle plane at a time whilst the signal is detected in a perpendicular(z-) direction. In example implementations of the 3D-IFC system 550, a1000 mW 488 nm laser (Coherent) that has a circular beam shape withGaussian energy distribution was used, which is collimated, expanded,and then focused to illuminates an area of 0.6 μm (z-direction) by 250μm (y-direction). A cylindrical lens and a 50×/0.55 objective lens(Mitutoyo) was used to form light sheet. An acousto-optic deflector(AOD) is configured in the optical system to produce high-speed scanningin z-direction (e.g., 200 kHz in this implementation).

-   -   ∂, which is determined by the field of view in x-direction D_(x)        the field of view in y-direction D_(y) and number of pinholes X,        ∂=tan⁻¹(D _(x)/(X·D _(y))). Also, because the z-direction        illumination light-sheet scans over a range that is larger than        the cell size, and at a speed that is much higher, typically        more than 20 times higher than the cell travelling speed in        y-direction, when cell passing one

$\begin{matrix}{{{\left. {\left. {{S(t)} = {\int{\int{\int\left\{ {{\int{\int{{C\left( {x^{\prime},{y^{\prime} - {v_{C}t}},z} \right)}{I\left( {z,t} \right)}{{psf}\left( {x - x^{\prime}} \right)}}}},{y - y^{\prime}}} \right.}}}} \right){dx}^{\prime}{dy}^{\prime}} \right\} \cdot {F\left( {{Mx},{My}} \right)}}{dxdydz}} = {\int{\int{\int{{\left\{ {{C\left( {x,{y - {v_{C}t}},z} \right)}{I\left( {z,t} \right)}} \right\} \cdot {F\left( {{Mx},{My}} \right)}}{dxdydz}}}}}} & (6)\end{matrix}$

where C(x, y, z) is the 3D cell fluorescence or scattering lightintensity profile, I(z,t) is the light-sheet illumination, F (x, y) isthe characteristic function of the spatial filter, v_(C) is the cellflowing speed, M is the magnification factor of the detection system.

The acoustic frequency sent to the acoustic transducer in the AOD may bevaried to deflect the beam to create illumination at differentz-position. The tuning voltage that produce continuous change ofacoustic frequency can be generated by various types of waveforms, suchas sinusoidal, triangle, etc. For the most laser power efficient, thetuning voltage is set to be changing in a sawtooth manner, so theposition of the light sheet in z-direction z₀(t) can be described as:

z ₀(tΕ(nT,(n+1)T))=v _(i)(t−nT)

n=0,1,2, . . .  (7)

where T is the light-sheet illumination scanning period, v_(i) is thescanning speed in z-direction.

By using the cylindrical lens, the laser is diverged to form alight-sheet with a height in y-direction of 200-400 μm. The scanninglight-sheet illumination I(z,t) can be described as Gaussian beam:

$\begin{matrix}{{I\left( {z,t} \right)} = {k \cdot e^{- \frac{{({z - {z_{0}(t)}})}^{2}}{\sigma^{2}}}}} & (8)\end{matrix}$

With oversampling PMT signal readouts, the spatial resolution inz-direction is diffraction limited. The Gaussian beam waist was measured0.73 μm and was approximated

I(z,t)≈k·δ(z−z ₀(t))  (9)

Two examples of the spatial filter are shown in FIG. 10.

FIG. 10 shows diagrams of examples for a spatial filter design. Twoexamples of spatial filters were placed at image plane. The top two andbottom two long slits with dimensions of 10 μm by 200 μm are for speeddetection. The other pinholes on the spatial filter are 10 μm by 20 μm(left) and 10 μm by 10 μm (right), for 3D image capturing with voxelsize of 2 μm and 1 μm in x-direction, respectively. The arrangement ofpinholes aligns the laser beam-waist.

Putting the slits used for speed detection aside, the characteristicfunction F(x, y) of the spatial filter is designed to be

$\begin{matrix}{{F\left( {x,y} \right)} = {\sum\limits_{q = 1}^{N}{{\delta\left( {x - x_{q}} \right)} \cdot {\delta\left( {y - y_{q}} \right)}}}} & (10)\end{matrix}$

where q=1, 2, . . . , Nis the number of pinholes on the spatial filter.The size of the pinhole, together with the NA of detection objectivelens, the cell flowing speed, and signal sampling rate determine thespatial resolution in x- and y-directions.

In the example implementations, the spatial filters were fabricatedusing electron beam lithography and the blackout area is made ofchromium with a thickness of 250 nm.

With the approximations above, when y_(q+1)−y_(q) is larger than cellsize (diameter), and cell projection is overlapped with j-th pinhole,

$\begin{matrix}\begin{matrix}{{S(t)} = {\int{{C\left( {x,{y - {{Mv}_{C}t}},{z_{0}(t)}} \right)}{\delta\left( {x - x_{q}} \right)}{\delta\left( {y - y_{q}} \right)}{dxdy}}}} \\{= {C\left( {x_{j},{y_{j} - {{Mv}_{C}t}},{z_{0}(t)}} \right)}}\end{matrix} & (11)\end{matrix}$

Using a 10×/0.28 detection objective lens and presuming cell is withinthe depth of field, and the example system PSF at multiple z was notconsidered. The light intensity signal detected by PMT (Hamamatsu) wasfirst amplified by an amplifier (Hamamatsu) with a bandwidth from DC to150 MHz, and then digitized by a digitizer (ADVANTECH) with a maximumsampling rate of 125 MS/s per channel.

Consequently, this example approach maps the 3D cell image into thetime-domain light intensity on a one-to-one basis. The 3D imageconstruction algorithm was written in MATLAB according to the equationsabove. Due to slight variance in flowing speed v_(C) of cells, theoriginal size of the 3D image of each cell can be slightly different. InThe example implementations, the original 3D image was then resized to100×100×100 pixels. 3D image batch processing was performed in ImageJ.

FIG. 11 shows another example embodiment of a 3D-IFC system, labeled1150, in accordance with some embodiments of the 3D-IFC system 550. The3D-IFC system 1150 includes an additional detection path, e.g.,providing a 2D transmission imaging mode. As shown in the diagram, theshadowed part (bottom right portion of the diagram) depicts thecomponents of the transmission image detection; which includes a spatialfilter 1171 for transmission detection having single slit (e.g., withdimensions of 50 μm by 2 mm), as well as a detection objective lens (DO)572 (e.g., 50×/0.55 detection objective lens), and photodetector 1172(e.g., PMT, photomultiplier tube), in data communication with the dataprocessing system (e.g., DIG 580).

Example methods for preparing the cells is described below.

Cell with Fluorescent Beads. In some example implementations, the humanembryonic kidney 293 (HEK-293) cells were cultured with complete culturemedia (DMEM, 10% Fetal Bovine Serum, 1% Penicillin Streptomycin) to 90%confluency in 10 cm petri dish. After 100× dilution of the 1.0 μmfluorescent beads (Ex/Em: 488/645 nm, e.g., T8883, ThermoFisher) fromthe stock solution (2% solids), 100 μL of the diluted solution was mixedwith 10 mL fresh cell culturing media and added to cell culturing plate.After continuous culturing for 10 hours, the HEK-293 cells wereharvested and stained with CellTrace CFSE cell proliferation kit (Ex/Em:492/517 nm, e.g., C34554, ThermoFisher) at a working concentration of 20μM. After the staining process, cells were fixed by 4% paraformaldehyde,washed and resuspended in 1× phosphate buffered saline (PBS). Beforeevery imaging experiment, the cell suspension was diluted in PBS to aconcentration of 1000 cells/μL.

CFSE Staining. In some example implementations, the HEK-293 cells may becultured with complete culture media to 98% confluency in 10 cm petridish, and then were harvested and resuspended to a concentration of1×10⁶ cells/mL in 1× PBS. The CFSE Cell Proliferation Kit (Ex/Em 492/517nm, e.g., C34554, Thermo Fisher) were added to the cell suspension at aworking concentration of 20 μM. After incubating the cells at 37° C. for30 minutes, fresh culture media (DMEM) were used to quench the stainingprocess and the HEK-293 cells were washed by 1× PBS and fixed by 4%paraformaldehyde. The fixed cells were washed and resuspended in 1× PBS.Protocol was also applied to stain CMK3 cells and human bloodleukocytes.

CMK3 Cell Irradiation Treatment and Immunostaining. In some exampleimplementations, the human glioblastoma CMK3 cells were cultured withcomplete culture media (DMEM-F12, 2% B27 supplement, 1% PenicillinStreptomycin, 1% Glutamax, 100 μg/L EGF, 100 μg/L FGF, 0.24% Heparin) inculture plates. The cells were harvested and resuspended to aconcentration of 1×10⁶ cells/mL in 1×PBS, and then stained with CFSE. Toinduce DNA double-strand breaks (DSB), CMK3 cells are treated with 6 Gyirradiation by cesium source irradiator. The treated cells were washedonce with 1×PBS and fixed with 1% paraformaldehyde 30 minutes postirradiation. The fixed cells were washed with PBS twice. Then 70%ethanol was added to the cells and the cells were incubated on ice for 1hour. After ethanol treatment, cells were washed with PBS twice andincubated in 1% TritonX-100 at room temperature for 10 minutes. Then,cells were washed with PBS once and incubated in 5% Bovine Serum Albumin(BSA) in PBS for 30 minutes at room temperature on shaker. Cells werethen washed with PBS once and incubated in anti-phospho-histone H2A.X(Ser139) Antibody, clone JBW301 at 1:300 dilution on ice on shaker for 1hour. After the primary antibody treatment, cells were washed twice with5% BSA and incubated in PerCP/Cy5.5 anti-mouse IgG1 Antibody at 1:100dilution on ice on shaker for 1 hour. At last, the stained cells werewashed twice with 5% BSA and resuspended in 1:3 diluted stabilizingfixative buffer in MilliQ water. Before every imaging experiment, thecells ware diluted in PBS to a concentration of 500 cells/μL.

Leukocytes Separation and staining. In some example implementations, thefresh human whole blood was harvested in EDTA tube from San Diego BloodBank. The red blood cells in the whole blood were first lysed by RBClysis buffer (00-4300-54, Invitrogen) and the leukocytes population washarvested by soft centrifuge after the lysing process. The harvestedleukocytes were then washed and resuspended to a concentration of 1×10⁶cells/mL in 1× PBS. The resuspended leukocytes were stained with CFSE.After the staining process, the cells were fixed by 4% paraformaldehyde,washed and resuspended in 1×PBS. Before every imaging experiment, thecell suspension was diluted in PBS to a concentration of 500 cells/μL.

As demonstrated by the example data, the disclosed 3D-IFC system is ableto provide an unprecedented access to high-throughput 3D image captureand analysis. The example implementations of the 3D-IFC system 550featured a cell flow speed of 0.2 m s⁻¹, a spatial resolution of 1 μm inthee axes, a field of view of 20 μm in all axes, and a throughput of 500cells per second. Some embodiments have the flexibility to directlyincrease the spatial resolution, field of view and 3D image capture ratethrough the change of spatial filter and the use of higher flow rate.Its information-rich 3D dark-field (side-scattering) image detection,coupled with 2D transmission image, offers possibilities for label-freeassays.

Example applications of the disclosed systems, methods and devicesinclude, but are not limited to, asymmetric division of T-cells intoeffector and memory cells, the secretory pathway of B cells, phenotypedrug discovery, protein or receptor translocations, tracking oforganelle formation or trafficking, and chromosome structuralaberrations, where 3D orientation and polarity are important, and otherapplications. In some implementations, the disclosed systems, methodsand devices can be integrated with image-based sorting functionality.

EXAMPLES

In some embodiments in accordance with the present technology (exampleA1), a three-dimensional imaging flow cytometry system includes aparticle flow system; a scanning light-sheet illumination systeminterfaced with the particle flow system; an optical detection systeminterfaced with the particle flow system; and a data processing systemin communication with the optical detection system and/or scanninglight-sheet illumination system. The particle flow system includes afluidic channel with clear optical paths carrying a fluid samplecontaining a particle; and a flow focusing system to produce confinedsample fluid to a fine stream in the fluidic channel. The scanninglight-sheet illumination system includes a light source to illuminatethe particle flowing in the fluidic channel; a device to redirect anillumination beam to different angles, which is programmable to generateperiodic angle change at high speed; and a cylindrical lens and/or otheroptical components to produce a structured light-sheet illumination thatallows optical scanning of illuminated sections of the particle at atime, such as a thin slice or a thin line of the particle. The opticaldetection system includes one or more photodetectors and a spatialfilter positioned at the image plane of the one or more photodetectorsto selectively allow light from specific position of the particle topass through and be detected by the one or more photodetectors. The dataprocessing system is configured to process the detected data and producea three-dimensional constructed image of the particle.

Example A2 includes the system of any of examples A1 or A3-A7, in whichthe optical detection system is operable to collect and detectfluorescence and/or scattering light.

Example A3 includes the system of any of examples A1-A2 or A4-A7, inwhich the optical detection system is configured to be placed at anyangle that the fluidic channel has an optically clear path.

Example A4 includes the system of any of examples A1-A3 or A5-A7, inwhich the optical detection system is operable to detect 1D optical datapoints in time of a 3D objection based on combination of (a) flow motionof the particle, (b) a scanning light-sheet illumination, and (c)spatially mapped filter to detect light intensity (e.g., includingfluorescence and scattering) from every position of the particle in asequentially-timed manner, such that the time-domain signal recorded bythe one or more photodetectors has a one-to-one relation from space totime.

Example A5 includes the system of any of examples A1-A4 or A6-A7, inwhich the data processing system includes a data streaming unit incommunication with the optical detection system.

Example A6 includes the system of any of examples A1-A5 or A7, in whichthe data processing system includes an image reconstruction algorithmthat includes specifications of illumination and detection protocols andcommands the scanning light-sheet illumination system and the opticaldetection system accordingly.

Example A7 includes the system of any of examples A1-A6, in which thedata processing system includes an image processing unit to process theimage data to determine properties associated with the particle.

In some embodiments in accordance with the present technology (exampleB1), an optical imaging system for three dimensional imaging in a flowcytometry system includes a light source to illuminate a particleflowing in a flow channel; one or more lenses configured above a regionof the flow channel; an acousto-optic deflector configured in an opticalpath of the optical photodetector and light source to provide high-speedscanning in a z-direction; and a single-element photodetector to detectfluorescence passed a spatial filter in each channel, in which the lightsource is configured to illuminate the particle flowing in the flowchannel in a single plane at a time while a signal is detected in aperpendicular direction.

Example B2 includes the optical imaging system of example B1, whereinthe system is configured to obtain optical signal data to reconstruct animage from time-domain detector output based on a combination of ascanned light sheet, the flow motion of the particle, and the spatialfilter, such that the photodetector detects an individual point in theparticle at a time, thereby allowing spatial-to-temporal transformation.

Example B3 includes the optical imaging system of example B1, whereinthe light source includes a laser.

In some embodiments in accordance with the present technology (exampleB4), a three-dimensional imaging flow cytometry system includes aparticle flow device structured to include a substrate, a channel formedon the substrate operable to flow particles including living cells alonga flow direction of the channel; an imaging system interfaced with theparticle flow device and operable to obtain image data in threedimensions associated with a particle during flow through the channel;and a data processing and control unit in communication with the imagingsystem, the data processing and control unit including a processorconfigured to process the image data obtained by the imaging system todetermine one or more properties associated with the cell from theprocessed image data, in which the imaging system is configured in alight sheet fluorescence microscopy configuration, which illuminates aspecimen in a single plane at a time whilst the signal is detected in aperpendicular direction.

Example B5 includes the three-dimensional imaging flow cytometry systemof example B4, wherein the imaging system includes the optical imagingsystem of any of examples B1-B3.

Example B6 includes the three-dimensional imaging flow cytometry systemof example B4, wherein the imaging system includes one or more lightsources to provide an input light at a region of the particle flowdevice, and an optical photodetector to capture the image data from theparticle illuminated by the input light.

Example B7 includes the three-dimensional imaging flow cytometry systemof example B6, wherein the optical photodetector includes an objectivelens of a microscope optically coupled to a spatial filter, an emissionfilter, and a photomultiplier tube.

Example B8 includes the three-dimensional imaging flow cytometry systemof example B7, wherein the optical photodetector further includes one ormore light guide elements to direct the input light at the first region,to direct light emitted or scattered by the cell to an optical elementof the optical photodetector, or both.

Example B9 includes the three-dimensional imaging flow cytometry systemof example B8, wherein the light guide element includes a dichroicmirror.

Example B10 includes the three-dimensional imaging flow cytometry systemof example B8, wherein the optical photodetector includes two or morephotomultiplier tubes to generate two or more corresponding signalsbased on two or more bands or types of light emitted or scattered by thecell.

In some embodiments in accordance with the present technology (exampleC1), a three-dimensional (3D) imaging flow cytometry system includes aparticle flow device including a substrate and a channel formed on thesubstrate to allow particles in a fluid to flow along a flow directionof the channel; an optical illumination system to produce atwo-dimensional (2D) sheet of light that scans over a plurality ofsections of a particle in the fluid, one section at a time, while theparticle is flowing along the flow direction in the channel, the opticalillumination system including a light source to produce a light beamthat is optically coupled to a light-sheet generation unit to modify thelight beam into the 2D sheet of light directed at the channel, whereinthe light-sheet generation unit includes a light redirection device toredirect the light beam to different angles and a cylindrical lensplaced after the light redirection device in an optical path with thechannel; an optical detection system interfaced with the particle flowdevice and operable to obtain optical signal data in three dimensionsassociated with the particle during flow through the channel, whereinthe optical detection system includes one or more photodetectors and aspatial filter positioned at an image plane of the one or morephotodetectors, the spatial filter including a plurality of apertures toselectively allow a portion of the 2D sheet of light over a scannedsection of the particle to pass through the spatial filter and bedetected by the one or more photodetectors; and a data processing unitin communication with the optical detection system and the opticalillumination system, the data processing unit including a processorconfigured to process the optical signal data obtained by the opticaldetection system and produce image data from which a 3D image of theparticle can be constructed.

Example C2 includes the system of any of examples C1 or C3-C15, whereinthe 2D sheet of light is directed at the channel in a second directionperpendicular to the flow direction, such that a first scan of the 2Dsheet of light forms a first plane comprising the flow direction and thesecond direction; and wherein the image plane of the optical detectionsystem is arranged along a third direction perpendicular to the flowdirection and the second direction, such that a second scan of the 2Dsheet of light forms a second plane parallel with the first plane andvaried by a distance from the first plane along the third direction.

Example C3 includes the system of example C2, wherein the opticalillumination system is configured to scan, while the particle is flowingin the flow direction, a first section of the particle in the firstplane at a first time point and to scan a second section of the particlein the second plane at a second time point, thereby producing a temporalsignal that spatially maps to the particle.

Example C4 includes the system of example C2, wherein the apertures ofthe spatial filter are positioned along the second direction such thateach aperture is configured to filter the 2D sheet of light tocorrespond to a voxel of the particle.

Example C5 includes the system of any of examples C1-C4 or C6-C15,wherein the data processing unit is operable produce the 3D image of theparticle, voxel-by-voxel.

Example C6 includes the system of any of examples C1-05 or C7-C15,wherein the image data of the particle includes a spatial resolution ofat least 1 μm in three axes and a field of view of 20 um in the threeaxes.

Example C7 includes the system of any of examples C1-C6 or C8-C15,wherein a flow rate of the particles in the channel is at least 0.2 ms⁻¹, and wherein the system is operable to produce the image data of theparticles at a throughput of 500 particles per second.

Example C8 includes the system of any of examples C1-C7 or C9-C15,wherein the plurality of apertures of the spatial filter are arranged ina pattern, wherein each aperture of the pattern is evenly spaced alongthe flow direction and varies in its position along a second directionperpendicular to the flow direction with respect to another aperture ofthe pattern.

Example C9 includes the system of any of examples C1-C8 or C10-C15,wherein the light redirection device includes an acousto-optic deflector(AOD).

Example C10 includes the system of any of examples C1-C9 or C11-C15,wherein the light-sheet generation unit is programmable, by the dataprocessing unit, to generate a periodic angle change at a speed of atleast 200 kHz.

Example C11 includes the system of any of examples C1-C10 or C12-C15,wherein the one or more photodetectors includes a photomultiplier tube.

Example C12 includes the system of any of examples C1-C11 or C13-C15,wherein the optical detection system includes an objective lensoptically coupled to the spatial filter, and an emission filteroptically coupled between the spatial filter and the photodetector.

Example C13 includes the system of example C12, wherein the opticaldetection system further includes one or more light guide elements todirect the light out of the spatial filter at toward multiplephotodetectors.

Example C14 includes the system of example C13, wherein the opticaldetection system includes two or more photomultiplier tubes to generatetwo or more corresponding signals based on two or more bands or types oflight emitted or scattered by the cell.

Example C15 includes the system of any of examples C1-C14, wherein theparticles include living cells.

In some embodiments in accordance with the present technology (exampleC16), a method for three-dimensional (3D) imaging of moving particlesincludes moving a particle through a channel along a first direction;scanning a plurality of sections of the particle, section by section, bydirecting individual two-dimensional (2D) sheets of light atcorresponding regions of the channel while the particle is movingthrough the channel; spatially filtering portions of the 2D sheets oflight over the scanned sections of the particle to allow certain opticalpoint signals through a spatial filter to be detected; detecting thespatially-filtered optical point signals corresponding to individualvoxels in three dimensions mapped to time points of detection; andprocessing the optical point signals to produce image data of theparticle.

Example C17 includes the method of any of examples C16 or C18-C24,comprising modifying a light beam produced from a light source into theindividual 2D sheets of light.

Example C18 includes the method of example C17, wherein the scanning theplurality of sections of the particle includes redirecting the lightbeam to different angles through a cylindrical lens that modifies aredirected light beam into a 2D sheet of light.

Example C19 includes the method of any of examples C16-C18 or C20-C24,wherein a first individual 2D sheet of light is directed at the channelin a second direction perpendicular to the first direction, such that afirst scan of the 2D sheet of light forms a first plane comprising thefirst direction and the second direction; and wherein an image plane forthe detecting is arranged along a third direction perpendicular to thefirst direction and the second direction, such that a second scan of asecond individual 2D sheet of light forms a second plane parallel withthe first plane and varied by a distance from the first plane along thethird direction.

Example C20 includes the method of any of examples C16-C19 or C21-C24,comprising producing a 3D image of the particle based on avoxel-by-voxel construction from the produced image data.

Example C21 includes the method of any of examples C16-C20 or C22-C24,wherein the image data of the particle includes a spatial resolution ofat least 1 μm in three axes and a field of view of 20 μm in the threeaxes.

Example C22 includes the method of any of examples C16-C21 or C22-C24,wherein the image data of the particles is produced at a throughput of500 particles per second.

Example C23 includes the method of any of examples C16-C22 or C24,wherein the particles include living cells.

Example C24 includes the method of any of examples C16-C23 implementedon the system of any of examples C1-C15.

In some embodiments in accordance with the present technology (exampleD1), a system for three-dimensional (3D) imaging of moving particlesincludes a particle motion device including a substrate to allowparticles to move along a travel path in a first direction; an opticalillumination system to produce an asymmetric illumination area of lightin a region of the travel path of a particle that scans over a pluralityof sections of the particle at multiple time points while the particleis moving, the asymmetric illumination area of light comprising onedimension of illumination thinner than the other dimension ofillumination to form a shape like an illumination plane, the opticalillumination system including a light source to produce a light beamthat is optically coupled to light redirection device to modify thelight beam by redirecting the light beam to different angles to have theasymmetric illumination area of light directed at the travel path ofparticle motion; an optical detection system optically interfaced withthe particle motion device and operable to obtain optical signal dataassociated with different parts of the particle corresponding to theparticle's volume during motion in the travel path, wherein the opticaldetection system includes one or more photodetectors and a spatialfilter positioned between the particle motion device and the one or morephotodetectors, the spatial filter including a plurality of apertures toselectively allow a portion of the asymmetric illumination area of lightover a scanned section of the particle to pass through the spatialfilter and be detected by the one or more photodetectors; and a dataprocessing unit in communication with the optical detection system, thedata processing unit including a processor configured to process theoptical signal data obtained by the optical detection system and producedata including information indicative of 3D features of the particle.

Example D2 includes the system of any of examples D1 or D3-D20, whereinthe asymmetric illumination area of light is directed at the region ofthe travel path in a second direction perpendicular to the firstdirection, such that a first scan of the asymmetric illumination area oflight forms a first plane comprising the first direction and the seconddirection; and wherein the image plane of the optical detection systemis arranged along a third direction perpendicular to the first directionand the second direction, such that a second scan of the asymmetricillumination area of light forms a second plane parallel with the firstplane and varied by a distance from the first plane along the thirddirection.

Example D3 includes the system of example D2 or D4, wherein the opticalillumination system is configured to scan, while the particle is movingin the first direction, a first section of the particle in the firstplane at a first time point and to scan a second section of the particlein the second plane at a second time point, thereby producing a temporalsignal that spatially maps to the particle.

Example D4 includes the system of example D2 or D3, wherein theapertures of the spatial filter are positioned along the seconddirection such that each aperture is configured to filter the asymmetricillumination area of light to correspond to a voxel of the particle.

Example D5 includes the system of any of examples D1-D4 or D6-D20,wherein the data processing unit is operable produce a 3D image of theparticle, voxel-by-voxel.

Example D6 includes the system of example D5, wherein the 3D image dataof the particle includes a spatial resolution of at least 1 μm in threeaxes and a field of view of about 20 μm in the three axes.

Example D7 includes the system of any of examples D1-D6 or D8-D20,wherein the particle motion device includes a particle flow cellincluding a channel formed on the substrate to allow a fluid containingthe particles to flow through the channel as the travel path.

Example D8 includes the system of example D7, wherein a flow rate of theparticles in the channel is at least 0.2 m s⁻¹, and wherein the systemis operable to produce the data of the particles at a throughput of 500particles per second.

Example D9 includes the system of any of examples D1-D8 or D10-D20,wherein the particles are fixed to the substrate, and wherein theparticle motion device includes a positioning system to move thesubstrate with respect to the optical illumination system and theoptical detection system such that the particle is moved along thetravel path and brought into the region upon which the asymmetricillumination area of light is scanned.

Example D10 includes the system of any of examples D1-D9 or D11-D20,wherein the plurality of apertures of the spatial filter are arranged ina pattern, wherein each aperture of the pattern is spaced along thefirst direction and varies in its position along a second directionperpendicular to the first direction with respect to another aperture ofthe pattern.

Example D11 includes the system of any of examples D1-D10 or D12-D20,wherein the light redirection device includes an acousto-optic deflector(AOD).

Example D12 includes the system of example D11, wherein the AOD isprogrammable, by the data processing unit, to generate a periodic anglechange at a speed of at least 100 kHz.

Example D13 includes the system of any of examples D1-D12 or D14-D20,wherein the light redirection device includes a multiplexed array ofscanning mirrors and/or a multiplexed array of scanning gratings.

Example D14 includes the system of any of examples D1-D13 or D15-D20,wherein the light redirection device is optically coupled with one ormore passive optical components, selected from a group consisting of acylindrical lens, a lens, a prism, and a spatial filter, to shape theredirected light on the travel path.

Example D15 includes the system of any of examples D1-D14 or D16-D20,wherein the one or more photodetectors includes a photomultiplier tube.

Example D16 includes the system of any of examples D1-D15 or D17-D20,wherein the optical detection system further includes an objective lensoptically coupled with the light redirection device.

Example D17 includes the system of any of examples D1-D16 or D18-D20,wherein the optical detection system includes an objective lensoptically coupled to the spatial filter.

Example D18 includes the system of any of examples D1-D17 or D19-D20,wherein the optical detection system further includes one or more lightguide elements to direct the light out of the spatial filter towardmultiple photodetectors.

Example D19 includes the system of example D18, wherein the opticaldetection system further includes two or more photodetectors to generatetwo or more corresponding signals based on two or more bands or types oflight emitted or scattered by the cell.

Example D20 includes the system of any of examples D1-D19, wherein theparticles include living cells.

In some embodiments in accordance with the present technology (exampleD21), a method for three-dimensional (3D) imaging of moving particlesincludes moving a particle along a first direction; scanning a pluralityof sections of the particle, section by section, by directing individualasymmetric illumination areas of light at corresponding regions whilethe particle is moving; spatially filtering portions of the asymmetricillumination area of light over the scanned sections of the particle inmotion to allow certain optical signals corresponding to the particle'svolume through a spatial filter to be detected; and detecting thespatially-filtered optical signals to obtain individual voxels in threedimensions mapped to time points of detection.

Example D22 includes the method of any of examples D21 or D23-D32,comprising modifying a light beam produced from a light source into theindividual asymmetric illumination areas of light.

Example D23 includes the method of any of examples D21-D22 or D24-D32,wherein the scanning the plurality of sections of the particle includesredirecting the light beam to different angles and shaping theredirected light beam into the asymmetric illumination areas of light.

Example D24 includes the method of any of examples D21-D23 or D25-D32,wherein a first individual asymmetric illumination area of light isdirected at a travel path of the particle in a second directionperpendicular to the first direction, such that a first scan of thefirst individual asymmetric illumination area of light forms a firstplane comprising the first direction and the second direction; andwherein an image plane for the detecting is arranged along a thirddirection perpendicular to the first direction and the second direction,such that a second scan of a second individual asymmetric illuminationarea of light forms a second plane parallel with the first plane andvaried by a distance from the first plane along the third direction.

Example D25 includes the method of any of examples D21-D24 or D26-D32,further comprising processing the optical signals to produce dataincluding information indicative of 3D features of the particle.

Example D26 includes the method of any of examples D21-D25 or D27-D32,comprising producing a 3D image of the particle based on avoxel-by-voxel construction from the produced data.

Example D27 includes the method of any of examples D21-D26 or D28-D32,wherein the data of the particle includes a spatial resolution of atleast 1 μm in three axes and a field of view of 20 μm in the three axes.

Example D28 includes the method of any of examples D21-D27 or D29-D32,wherein the data of the particles is produced at a throughput of 500particles per second.

Example D29 includes the method of any of examples D21-D28 or D30-D32,wherein the particle is moved by a particle flow cell including achannel formed on a substrate to flow a fluid containing the particlethrough the channel along the first direction.

Example D30 includes the method of any of examples D21-D29 or D31-D32,wherein the particle is fixed to a substrate, and the substrate is movedby a positioning system, such that the particle moves along the firstdirection on the substrate.

Example D31 includes the method of any of example D21-D30 or D32,wherein the particles include living cells.

Example D32 includes the method of any of examples D21-D31 implementedon the system of any of examples D1-D20.

Implementations of the subject matter and the functional operationsdescribed in this patent document and attached appendices can beimplemented in various systems, digital electronic circuitry, or incomputer software, firmware, or hardware, including the structuresdisclosed in this specification and their structural equivalents, or incombinations of one or more of them. Implementations of the subjectmatter described in this specification can be implemented as one or morecomputer program products, i.e., one or more modules of computer programinstructions encoded on a tangible and non-transitory computer readablemedium for execution by, or to control the operation of, data processingapparatus. The computer readable medium can be a machine-readablestorage device, a machine-readable storage substrate, a memory device, acomposition of matter effecting a machine-readable propagated signal, ora combination of one or more of them. The term “data processing unit” or“data processing apparatus” encompasses all apparatus, devices, andmachines for processing data, including by way of example a programmableprocessor, a computer, or multiple processors or computers. Theapparatus can include, in addition to hardware, code that creates anexecution environment for the computer program in question, e.g., codethat constitutes processor firmware, a protocol stack, a databasemanagement system, an operating system, or a combination of one or moreof them.

A computer program (also known as a program, software, softwareapplication, script, or code) can be written in any form of programminglanguage, including compiled or interpreted languages, and it can bedeployed in any form, including as a stand-alone program or as a module,component, subroutine, or other unit suitable for use in a computingenvironment. A computer program does not necessarily correspond to afile in a file system. A program can be stored in a portion of a filethat holds other programs or data (e.g., one or more scripts stored in amarkup language document), in a single file dedicated to the program inquestion, or in multiple coordinated files (e.g., files that store oneor more modules, sub programs, or portions of code). A computer programcan be deployed to be executed on one computer or on multiple computersthat are located at one site or distributed across multiple sites andinterconnected by a communication network.

The processes and logic flows described in this specification can beperformed by one or more programmable processors executing one or morecomputer programs to perform functions by operating on input data andgenerating output. The processes and logic flows can also be performedby, and apparatus can also be implemented as, special purpose logiccircuitry, e.g., an FPGA (field programmable gate array) or an ASIC(application specific integrated circuit).

Processors suitable for the execution of a computer program include, byway of example, both general and special purpose microprocessors, andany one or more processors of any kind of digital computer. Generally, aprocessor will receive instructions and data from a read only memory ora random access memory or both. The essential elements of a computer area processor for performing instructions and one or more memory devicesfor storing instructions and data. Generally, a computer will alsoinclude, or be operatively coupled to receive data from or transfer datato, or both, one or more mass storage devices for storing data, e.g.,magnetic, magneto optical disks, or optical disks. However, a computerneed not have such devices. Computer readable media suitable for storingcomputer program instructions and data include all forms of nonvolatilememory, media and memory devices, including by way of examplesemiconductor memory devices, e.g., EPROM, EEPROM, and flash memorydevices. The processor and the memory can be supplemented by, orincorporated in, special purpose logic circuitry.

The term “about,” as used herein when referring to a measurable valuesuch as an amount or concentration and the like, is meant to encompassvariations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specifiedamount.

It is intended that the specification, together with the drawings, beconsidered exemplary only, where exemplary means an example. As usedherein, the singular forms “a”, “an” and “the” are intended to includethe plural forms as well, unless the context clearly indicatesotherwise. Additionally, the use of “or” is intended to include“and/or”, unless the context clearly indicates otherwise.

While this patent document and attached appendices contain manyspecifics, these should not be construed as limitations on the scope ofany invention or of what may be claimed, but rather as descriptions offeatures that may be specific to particular embodiments of particularinventions. Certain features that are described in this patent documentand attached appendices in the context of separate embodiments can alsobe implemented in combination in a single embodiment. Conversely,various features that are described in the context of a singleembodiment can also be implemented in multiple embodiments separately orin any suitable subcombination. Moreover, although features may bedescribed above as acting in certain combinations and even initiallyclaimed as such, one or more features from a claimed combination can insome cases be excised from the combination, and the claimed combinationmay be directed to a subcombination or variation of a subcombination.

Similarly, while operations are depicted in the drawings in a particularorder, this should not be understood as requiring that such operationsbe performed in the particular order shown or in sequential order, orthat all illustrated operations be performed, to achieve desirableresults. Moreover, the separation of various system components in theembodiments described in this patent document and attached appendicesshould not be understood as requiring such separation in allembodiments.

Only a few implementations and examples are described and otherimplementations, enhancements and variations can be made based on whatis described and illustrated in this patent document and attachedappendices.

What is claimed is:
 1. A system for imaging a moving particle,comprising: a light source emitting a light beam; a light redirectiondevice to modulate the light beam to produce an illumination area thatis repeatedly swept across an interrogation area where the movingparticle travels through, thereby producing an optical signal when theillumination area illuminates the moving particle; a spatial filter toallow portions of the optical signal to pass through, thereby generatinga filtered optical signal corresponding to a section of the movingparticle; and a light detection device for detecting the filteredoptical signal.
 2. The system of claim 1, wherein the light redirectiondevice comprises an acousto-optic deflector.
 3. The system of claim 2,wherein the acousto-optic deflector generates a periodic angle change ata rate of 100 kHz to 1 MHz.
 4. The system of claim 2, wherein theacousto-optic deflector produces a deflected first-order beam at adifferent angle for each frequency.
 5. The system of claim 2, whereinthe filtered optical signal detected by the light detection device issynchronized with a reference output of a tuning voltage of a driver forthe acoustic-optic deflector.
 6. The system of claim 1, wherein theillumination area comprises an asymmetric illumination area comprisingone dimension of illumination thinner than another dimension ofillumination to form a shape like an illumination plane.
 7. The systemof claim 6, wherein the illumination area is swept across theinterrogation area in a direction approximately perpendicular to theillumination plane.
 8. The system of claim 1, wherein the lightredirection device produces an asymmetric intensity profile.
 9. Thesystem of claim 1, wherein the interrogation area comprises a fluidicchannel of a flow cell.
 10. The system of claim 9, wherein the flow cellcomprises a sheath flow and sample flow.
 11. The system of claim 9,wherein the flow cell comprises a flow rate of about 10 centimeters persecond to 20 centimeters per second.
 12. The system of claim 1, whereinthe system is operable to produce image data of a plurality of movingparticles at a throughput of 500 particles per second.
 13. The system ofclaim 1, further comprising a particle motion device.
 14. The system ofclaim 13, wherein the particle motion device comprises a substrate,wherein a particle is fixed to the substrate and the substrate ismoveable by a positioning system, thereby providing the moving particle.15. The system of claim 1, wherein the filtered optical signal comprisesa one-dimensional data point corresponding to a time point.
 16. Thesystem of claim 15, wherein signals outside a specific area or aspecific time point are blocked by the spatial filter.
 17. The system ofclaim 1, wherein the optical illumination system is configured to scan,while the moving particle is moving in a first direction, a firstsection of the moving particle in a first plane at a first time pointand to scan a second section of the moving particle in a second plane ata second time point, thereby producing a temporal signal that spatiallymaps to the moving particle.
 18. The system of claim 1, furthercomprising a data processing unit in communication with the lightdetection device, wherein the light detection device converts thefiltered optical signal into filtered optical signal data, and whereinthe data processing unit includes a processor configured to process thefiltered optical signal data obtained by the light detection device andproduce data including information indicative of three dimensionalfeatures of the moving particle.
 19. The system of claim 1, wherein thespatial filter comprises a plurality of apertures.
 20. A method ofimaging a moving particle, comprising: emitting a light beam from alight source; modulating the light beam to produce an illumination area,wherein the modulating the light beam includes repeatedly sweeping theillumination area across an interrogation area which the moving particletravels through, thereby producing an optical signal when theillumination area illuminates the moving particle; filtering portions ofthe optical signal, thereby generating a filtered optical signalcorresponding to a section of the moving particle; and detecting thefiltered optical signal.
 21. The method of claim 20, further comprisingconstruing a volume of the moving particle from the filtered opticalsignal.
 22. A system for imaging a moving particle, comprising: a lightsource to emit a light beam, wherein the light beam illuminates anasymmetric area with a first dimension much wider than a seconddimension of the illuminated area; a light beam modulation device tomodify the light beam to illuminate different parts of the movingparticle at different times, thereby producing an optical signal relatedto the properties of the moving particle; a spatial filter comprised ofone or more apertures to allow portions of the optical signal to enteran optical detector, thereby generating a spatially filtered opticalsignal corresponding to a section of the moving particle; a lightdetection device to detect the spatially filtered optical signal andoutput a detected signal; and a signal processing unit to process thedetected signal and display the detected signal as an image of themoving particle.
 23. A system for imaging a moving particle, comprising:a light source to emit a light beam, wherein the light beam illuminatesan asymmetric area with a first dimension much wider than a seconddimension of the illuminated area; a light beam modulation device tomodify the light beam to illuminate different parts of the movingparticle at different times, thereby producing an optical signal relatedto properties of the moving particle; a spatial filter to allow portionsof the optical signal to enter a light detection device, therebygenerating a spatially filtered optical signal corresponding to asection of the moving particle; and a signal processing unit to apply analgorithm to transform a temporal signal from the light detection deviceinto an image of the moving particle.
 24. A method of imaging a movingparticle, comprising: emitting a light beam from a light source, whereinthe light beam illuminates an asymmetric area with a first dimensionmuch wider than a second dimension of the illuminated area; modulatingthe light beam to illuminate different parts of the moving particle atdifferent times, thereby producing an optical signal related to theproperties of the moving particle; filtering portions of the opticalsignal, thereby generating a filtered optical signal corresponding to asection of the moving particle; detecting the filtered optical signal;processing the filtered optical signal to generate a detected signal;and displaying the detected signal as an image of the moving particle.